Breast cancer is the most commonly diagnosed cancer in women and is a second leading cause of death among woman. Despite the significant progress in molecular understanding and personalized therapies of breast cancer, this disease in progressed metastatic stage remains incurable and treatment options available to patients provide only modest survival benefit. Efficacy of existing systemic chemotherapy is limited in delivery, access and penetration into solid tumors, as well as by toxicity to patients. Thus, to develop more effective and safer therapies of breast cancer, it is necessary to implement an efficient targeted drug delivery system. Caveolae at the surface of tumor endothelial cells act as a transendothelial transport system that actively pumps targeted antibodies from the blood circulation into the tumor tissue. Our findings support that targeting caveolae provides a platform for rapid pumping of targeted antibodies with attached cargo specifically into breast tumors at concentrations greatly exceeding maximum levels in the blood. Using these recent findings, we intend to target antibody drug conjugates (ADC) of DM1 maytansinoid, one of the most potent anticancer drugs to target truncated form of Annexin A1 in tumor endothelial caveolae in order to deliver this powerful therapeutic effectively inside of breast tumors. We have demonstrated that AnnA1 is expressed in the vasculature and caveolae of human tumors. Recently, we have generated humanized antibody against AnnA1 (hAnnA1) and our preliminary experiments demonstrate strong therapeutic response of breast cancer to hAnnA1 conjugated to DM1 (hAnnA1-DM1 ADC). We hypothesize that by pumping targeted antibodies armed with DM1 drug into breast tumors, caveolae can rapidly and specifically accumulate this therapeutic agent inside tumors, leading to tumor destruction with significantly reduced toxicity. We propose these Specific Aims: Aim 1. To generate and characterize ADCs. We will prepare series of conjugates with DM1 drug covalently attached to hAnnA1 antibody using non-cleavable and cleavable linkers. Based on the screening in Aim2, we will generate THIOMAB hAnnA1 with select linker chemistry. Aim 2. Screening for optimal linker chemistry, production, validation and pharmacokinetic analysis of tumor vascular caveolae-targeted DM1 THIOMAB ADC in rodent tumor model of breast cancer. These studies will enable implementing optimal linker chemistry to develop ADC with site-specific attachment of DM1. Aim 3. Evaluation of therapeutic efficacy of the tumor vascular caveolae-targeted DM1 ADCs in clinically-relevant models of breast cancer. Therapeutic studies in clinically-relevant orthotopic models of breast cancer, including intravital microscopy model of human tumor spheroids engrafted into human breast tissue, and patient-derived breast cancer xenografts, including triple-negative and HER2+ models, will provide platform to evaluate novel hAnnA1-DM1 ADC therapy. The proposed project will help to establish framework for clinical translation of tumor-caveolae targeted ADC for treatment of breast cancer.